Biochemical binding assays are widely used to determine the presence and the concentration of analytes in biological specimens. Such assays are based on the concept of binding partners. An analyte of interest binds to an analyte binding agent (such as, for example, an antibody to the analyte, or a receptor for the analyte), and the analyte and the analyte binding agent are thus referred to as “binding partners”. When one of the binding partners is bound to a solid phase, the assay is referred to as a heterogeneous assay. Such heterogeneous assays include, for example, the sandwich method, the indirect method, and the competitive method, all terms readily recognized in the art.
The sensitivity of an assay typically refers to the smallest mass of analyte that generates a statistically significant change in the signal generated by the assay when compared to the signal reading obtained in the absence of the analyte. Increased sensitivity is desirable because it permits detection of smaller amounts of analyte as well as an overall higher precision measurement of an analyte.
Non-specific binding refers to non-specific interactions of the binding partners in a heterogeneous assay system with a solid phase. Non-specific binding often reduces the sensitivity of heterogeneous assays, and it is therefore desirable to reduce such non-specific binding.
A number of methods are known for this purpose. For example, proteins, such as bovine serum albumin (BSA), gelatin, and casein, have been added to assay reagents or preadsorbed on the solid phase in order to block non-specific adsorption sites. Additionally, the use of various surfactants, often in high concentration, has been reported in the literature.
While these techniques may assist in reducing some non-specific adsorption, many of the techniques have been associated with interference with the desired specific interaction of the binding partners. These techniques may also lead to the displacement of the complex which is formed between the binding partners. Additionally, despite the use of high concentrations of protein and surfactant, a considerable amount of non-specific binding typically still exists in many heterogeneous assays. Alternative means to reduce non-specific binding in heterogeneous assays are thus needed.
This is especially true in the case of assays for cardiac troponin I where the levels of analyte being detected are very small and increased sensitivity is necessary for accurate and useful assay results. Cardiac Troponin I measurement aids in the accurate diagnosis of acute myocardial infarction and in the risk stratification of patients with non-ST-segment elevation acute coronary syndromes with respect to relative risk of mortality, myocardial infarction, or increased probability of ischemic events requiring urgent revascularization procedures.
Troponin I (TnI) is a protein normally found in muscle tissue that, in conjunction with Troponin T and Troponin C, regulates the calcium dependent interaction of actin and myosin (Tobacman, Annu Rev Physiol 58:447-481, 1996). Three isotypes of TnI have been identified: one associated with fast-twitch skeletal muscle, one with slow-twitch skeletal muscle, and one with cardiac muscle (Wilkinson and Grand, Nature 271:31-35, 1978; Bodor, J Clin Immunoassay 17(1):40-44, 1994). The cardiac form has an additional 31 amino acid residues at the N-terminus and is the only troponin isoform present in the myocardium (Vallins et al., FEBS Letts 270(1,2):57-61, 1990) Clinical studies have demonstrated that cardiac troponin I (cTnI) is detectable in the bloodstream 4-6 hours after an acute myocardial infarct (AMI) and remains elevated for several days thereafter (Mair et al., Clin Chem 41(9):1266-1272, 1995; Larue et al., Clin Chem 39(6):972-979, 1993). Thus, cTnI elevation covers the diagnostic windows of both creatine kinase-MB (CK-MB) and lactate dehydrogenase (Bodor, J Clin Immunoassay 17(1):40-44, 1994). Further studies have indicated that cTnI has a higher clinical specificity for myocardial injury than does CK-MB (Adams et al., Circulation 88(1):101-106, 1993; Apple et al., Clin Chim Acta 237:59-66, 1995).
Because of its cardiac specificity and sensitivity, cTnI has been used as a reliable marker in evaluating patients with unstable angina and non-ST segment elevation acute coronary syndrome (ACS). Previous clinical studies of patients with ACS (Lindahl et al., J Am Coll Cardiol 38:1497-1498, 2001; Venge et al., Am J Cardiol 89:1035-1041, 2002) have shown that minor increases in cTnI values provide important prognostic information about the short and long term risk of death (Galvani et al., Circulation 95:2053-2059, 1997; Antman et al., N Eng J Med 335:1342-1349, 1996; Ottani et al., Am Heart J40:917-927, 2000; Heidenreich et al., J Am Coll Cardiol 38:478-485, 2001). Ultimately, the assessment of the prognosis can be useful in identifying patients most likely to benefit from specific therapeutic interventions.
Thus, any reagents and methods for reducing non-specific binding in heterogeneous assays for cTnI, thus leading to increased sensitivity of cTnI assays, are desirable.